Aneuploidy generates enhanced nucleotide dependency and sensitivity to metabolic perturbation.

Despite the general detriment of aneuploidy to cellular fitness, >90% of solid tumors carry an imbalanced karyotype. This existing paradox and the molecular responses to aneuploidy remain poorly understood. Here, we explore these cellular stresses and unique vulnerabilities of aneuploidy in human mammary epithelial cells (HMECs) enriched for breast cancer-associated copy number alterations (CNAs). To uncover the genetic dependencies specific to aneuploid cells, we conducted a comprehensive, genome-wide CRISPR knockout screen in isogenic aneuploid and diploid HMEC lines. Our study reveals that aneuploid HMECs exhibit an increased reliance on pyrimidine biosynthesis and mitochondrial oxidative phosphorylation genes and demonstrate heightened fitness advantages upon loss of tumor suppressor genes. Using an integrative multiomic analysis, we confirmed nucleotide pool insufficiency as a key contributor to widespread cellular dysfunction in aneuploid HMECs with net copy number gain. Although diploid cells can switch seamlessly between pyrimidine synthesis and salvage, cells with increased chromosomal content exhibit p53 activation and S-phase arrest when relying on salvage alone, alongside increased sensitivity to DNA-damaging chemotherapeutics. This work advances our understanding of the consequences of aneuploidy and uncovers potential avenues for patient stratification and therapeutic intervention based on tumor ploidy.